Magnetic cells for localizing delivery and tissue repair

ABSTRACT

Normal or genetically modified cell(s) having magnetic nanoparticle(s) bound (affixed) to their surfaces and methods of delivery to target tissues, e.g. for treatment of disease and or injury.

The invention described herein was developed in part using funds from agrant from the Government of the United States of America (NEI/NIH grantno. R21-017971). The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to magnetic cell(s), and methods of use. The cellsare either naturally occurring or genetically altered animal somatic orstem cell(s), generated either from a patient/individual or from aliving or deceased donor. Magnetic particles are affixed to the outersurface of the cell. The magnetic cell(s) can then be administered to asubject, e.g. by injection or implantation, and their localizationcontrolled by application of an external magnetic field, e.g. a magnetpositioned outside the body. This allows the cells to be magneticallydirected to specific sites in the body, wherein the cells themselves, orvarious contents thereof such as enzymes, hormones, transmitters, andthe like, will be useful for investigational, diagnostic or therapeuticpurposes.

BACKGROUND

A large number of diseases and disorders result, from the dysfunction ofa specific tissue or organ. A number of these are currently treated bytransplantation, e.g. heart transplantation for certain types of cardiacdysfunction, corneal transplantation for endothelial cell dysfunction,stem cells for retinal neuroprotection, etc. However, transplantationprocedures are invasive, have varying rates of success, and are not yeteven available for many types of diseases or disorders, in particularfor a number of eye diseases, for example, including diseases of thecornea (including but not limited to endothelial or stromaldystrophies), diseases of retinal ganglion cells and the optic nerve(including but not limited to glaucoma, ischemic optic neuropathies,other optic neuropathies), and diseases of retinal photoreceptors andretinal pigment epithelium (including but not limited to Leber'scongenital amaurosis, retinitis pigmentosa and age-related maculardegeneration)

Although in many cases it would seem desirable to administer new“healthy” cells, e.g. by injection or infusion, simply injecting suchcells generally does not work as they do not remain localized and stickto or become incorporated into the patients' tissue. For example,healthy corneal endothelial cells are inefficiently incorporated into apatient's diseased cornea when injected into the anterior chamber of theeye (e.g. Mimura et al, Invest. Oplrthalmol. Vis. Sci. 2005,46(10):3637-44), and healthy retinal ganglion cells are not incorporatedinto the retina when injected into the vitreous cavity of the eye. Mostcurrent technology depends on whole tissue transplants, or in the caseof stem cell clinical trials, there are no techniques for controllingthe cells' localization in vivo. A stem cell transplantation clinicaltrial for retinitis pigmentosa, for example, uses subretinal injectionof hematopoetic stem cells, but does nothing to control theirlocalization there, or keep them from floating or migrating away aftersurgical implantation. As another example, corneal endothelial cellsinjected into the anterior chamber of the eye will simply fall bygravity away from the cornea, and not properly attach. Thus, thereremains a need for new methods for targeting cells to specific tissuesfor therapeutic purposes.

Consigny (U.S. Pat. No. 6,203,487) described a method in which magneticparticles of micron size are inserted into cells for the purpose offocalized delivery. In the present application, a method of attachingmagnetic nanoparticles with diameters of 500 nm or less to the outersurface of cells and delivering them to a target tissue is described.This is advantageous in many applications (e.g. applications involvingthe eye), because smaller particles that are not internalized into cellscan be degraded from the cell surface and easily excreted after thecells have been situated. For example, in applications involving theeye, the particles can be excreted without clogging ocular outflow andthereby raising intraocular pressure.

SUMMARY

It is an object to provide a method for delivery of specific cells to atarget tissue of a subject by attaching or affixing magnetic particlesto the outer surface of said cells, administering the magneticparticle-comprising cell to the subject, and applying a magnetic fieldto said target tissue under conditions such that said magneticparticle-comprising cell is delivered to the target tissue. The coatedparticles and cells comprising them are useful, for example, forprevention or treatment of diseases and disorders.

Accordingly, the invention provides magnetized cells comprising coatedmagnetic nanoparticles, methods of obtaining the magnetized cells,methods of delivering them to target tissues, and methods of prophylaxisand/or treatment of diseases and disorders using the magnetized cells.

Specific cells for magnetization by attachment of particles and deliveryinclude, e.g., neurons, neuroglial cells, endothelial cells,fibroblasts, smooth or skeletal muscle cells, epithelial cells,pancreatic islet cells, hepatocytes, schwann cells, dermal cells, kidneycells, bladder cells, cartilage cells, or bone cells. For example,relevant to the eye and visual system, ocular cells, optic nerve cells,corneal cells, whether epithelial, stromal, or endothelial; a retinalcell, whether a retinal neuron such as a retinal ganglion cell orphotoreceptor or other retinal neuron, or a retinal glial cell whether aretinal muller glial cell or retinal astrocyte or other, or a retinalendothelial cell or pericyte; a retinal progenitor cell; a retinal stemcell; an optic nerve glial cell whether an astrocyte, oligodendrocyte,microglial cell, or one of their precursors; or another stem orprogenitor cell capable of either differentiating into an ocular oroptic nerve cell or capable of supporting the survival or growth ornormal function of an ocular or optic nerve cell are delivered tospecific areas of the eye. Cells may be normal or genetically modified,e.g. by insertion of particular gene(s) or gene fragments that may beconsidered advantageous for the condition being treated. For specificapplications, at least one, but typically many nanoparticles will beaffixed or bound to an individual cell.

The term “subject” is intended to mean an animal, for example a mammal,in particular a human.

The term “magnetic nanoparticle” is intended to mean a particle of nolarger than about 500 nm, more usually no larger than 200 nm, havingmagnetic properties. Particles that can be used include nanospheres,conjugates, micelles, colloids, aggregates and complexes comprisingferromagnetic, paramagnetic or superparamagnetic material, such as iron,nickel, cobalt, and alloys thereof, as suitable for in vivo use. Forexample, the magnetic nanoparticles may comprise iron in anyferromagnetic form, with or without an inert surface coating, with itssurface chemically modified to allow the binding of an antibody, orantibody fragment, or protein or sugar fragment that binds to cells.Persons of skill in the art will appreciate that compounds havingexcessive toxicity when used according to the method are to be avoided.It is expected that in many applications the particles will be absorbedand excreted over time, and that small amounts of otherwise toxicparticles may accordingly not present a problem when used in the method.

In general, it is expected that the magnetic nanoparticles will have amean diameter of between 5 and 500 nm, more particularly between 40 and400 nm, most particularly between 40 and 100 nm, with a standarddeviation of 50% or lower, more usually 20% or lower. Difficulties inusing nanoparticles over micron-scale particles include particleaggregation, particle tracking and observation, and ability to mobilizethe particles by external magnetic fields, all of which are considerablyeasier when using micron-scale or larger particles. For this reason,previous efforts likely focused on micron-scale particles and ignoredthe possible advantages of nanoparticles. We have overcome a number ofthese difficulties, and have now discovered a number of advantages ofusing nanoparticles over micron-scale particles. Advantages includeability to bind to cell surfaces without stimulating endocytosis;ability to be shed from cell surfaces or, when internalized, excretedfrom cells; and ability to be excreted from the eye or the body whenshed from cells.

Coatings to be affixed to the particles include non-specific bindingagents such as inert metals like gold or dextrans or polymers; and/orspecific binding agents such as antibodies that are specific tocell-surface antigens. For example, antibodies directed against SSEA-1bind to many types of stern cells, and nanoparticles coated withanti-SSEA-1 antibodies can be used to convert stern cells into magneticstem cells. Similarly, many cells express specific integrin receptors,and antibodies against these integrin receptors bound to magneticnanoparticles bind to specific cells such as corneal endothelial cellsand create magnetic corneal endothelial cells.

Coatings are affixed to the particles by standard methods used broadlyin the field (for example, see (Schroder et al., 1986; Douglas et al.,1987; Sestier et al., 1998; Perrin et al., 1999; McCloskey et al., 2000;Tibbe et al., 2001).

By “target tissue” is meant any specific tissue type or location, e.g.within an organ or tissue, to which it is desirable to deliver thecells. For example, ocular cells or other stem cells may be delivered tothe eye, or a specific region of the eye, e.g. to the cornea, opticnerve, retina, etc.

The magnetic nanoparticles can be affixed to the surface of the cells byany effective means known to those of skill in the art. For example, themagnetic particle may be affixed to the cell by means of an antibody,e.g. an antibody specific for a surface antigen present on the cell.Surface coatings comprising, for example, anti-L1, anti-trkB,anti-integrin, and cholera toxin subunit B may be placed on the magneticparticles for the purpose of attaching them to retinal ganglion cells.The magnetic particle may also be affixed to the cell using a specificligand for which a receptor is present on the cell. For example, themagnetic particles can be functionalized for attachment to retinalganglion cells (RGCs) using brain-derived neurotrophic factor (BDNF).Other means of attaching the particles to the cells include non-specificchemical modifications such as carboxy or amide groups, or coatings ofsugars or dextrans, or coatings of polymers such as amino acids polymerslike poly-lysine, or coatings of otherwise inert coatings that bindcells. Coated magnetic nanoparticles will be affixed to the outersurface of cells by co-incubation in a general media that affordsadequate cell survival during the co-incubation period; the media is notgenerally found to be germane to the affixing process. In general, abalanced salt solution at physiologic pH around 7.4 will suffice;supplements to the media that enhance cell survival during the processare the topic of other published work specific to cell types being usedand are not germane to this invention. Co-incubation time andtemperature may depend on the specific cell type being converted into amagnetic cell; for example, binding to retinal ganglion cells usingmagnetic nanoparticles coated with an anti-TrkB antibody occursmaximally after 4 hours at 37 degrees celcius, but may also be performedby co-incubation at 4° C. overnight. Excess magnetic nanoparticles notbound to cells can be washed away either by spinning the cells down in acentrifuge at a speed that pellets the cells but not the unboundmagnetic nanoparticles, or by eluting the magnetic nanoparticle-boundcells away from the unbound cells using a magnetic field, or both.

The magnetic nanoparticle comprising cells may be administered to asubject by any suitable means, e.g. by injection, infusion or surfaceapplication. The cells may be suspended in anypharmaceutically/physiologically acceptable medium or solution, such asfor example, isotonic saline solution or culture medium suitable for invivo delivery to a subject. Additional excipients and carriers may beadded as are found suitable by those of skill in the art. Suitablesolutions and delivery vehicles are described in Remington: The Scienceand Practice of Pharmacy, 21^(st) Ed. (2005). For some applications,such as delivery of replacement cells to the corneal endothelium forendothelial damage or dystrophy, or delivery of stem cells under theretina for age-related macular degeneration or retinitis pigmentosa,10³-10⁶ cells will be delivered by injection in a volume of 3-300 μL butmore typically around 10⁴ cells in a volume of 10-100 μL. In otherapplications, such as delivery of stem cells to enhance retinal ganglioncell survival in diseases like glaucoma or ischemic optic neuropathy,10³-10⁶ cells will be delivered by injection in a volume of 3-300 μL butmore typically around 10⁵ cells in a volume of 200 μL. In otherapplications, such as delivery of replacement liver cells in diseaseslike cirrhosis or hepatitis, 10⁶-10⁹ cells will be delivered byintravenous infusion in a volume of 1-500 mL but more typically around10⁷ cells in a volume of 200 mL. When considering the delivery of cellsmeant for carrying toxic compounds to specific tissues for example incancer therapeutics, the cell number will have to be carefully titratedagainst systemic toxicity to the patient.

Also provided are normal or genetically modified cell(s) having amagnetic nanoparticle bound or affixed to its surface covalently or byantibody-antigen linkage, in particular ocular cells such as cornealcell(s), whether epithelial, stromal, or endothelial; retinal cell(s),whether a retinal neuron such as a retinal ganglion cell orphotoreceptor or other retinal neuron, or retinal glial cell(s) whetherretinal muller glial cell(s) or retinal astrocyte(s) or other, orretinal endothelial cell(s) or pericyte(s); retinal progenitor cell(s);retinal stem cell(s); optic nerve glial cell(s) whether an astrocyte(s),oligodendrocyte(s), microglial cell(s), or their precursors; or otherstem or progenitor cell(s) capable of either differentiating into ocularor optic nerve cell(s) or supporting the survival or growth or normalfunction of an ocular or optic nerve cell. In one embodiment, themagnetic particle(s) are bound via an antibody-antigen complex. Ingeneral, the magnetic particle will have a diameter of less than 500 nm,preferably less than 200 nm, more preferably less than 100 nm. Themagnetic particle may comprise iron in any ferromagnetic form, with orwithout an inert surface coating, with its surface chemically modifiedto allow the binding of an antibody, or antibody fragment, or protein orsugar fragment that binds to cells. The particle may also comprise othermagnetic substances, such as nickel or cobalt.

Typically, the magnetic particle-comprising cells will be isolatedcells, that is, contained/maintained in an environment that is distinctfrom their natural state. For example, the cells may be grown in tissueculture, or isolated from a donor animal or a subject who is to betreated, and maintained under suitable in vitro conditions formodification and use.

Cells with magnetic nanoparticles so bound can be used to treat avariety of diseases and disorders of the eye, such as, for example,macular degeneration, retinitis pigmentosa, and other retinaldegenerations; glaucoma, ischemic optic neuropathy, and other retinalganglion cell and optic nerve degenerations; Fuch's endothelialdystrophy, pseudophakic bullous kerathopathy, and other cornealendothelial cell degenerations; corneal epithelial cell degeneration,corneal limbal cell deficiency, and other corneal epithelial celldegenerations; ocular melanoma, lymphoma, and other ocular cancers. Thecells comprising magnetic nanoparticles can also be used to treat otherdiseases and disorders wherein it may be desirable or advantageous totarget a specific tissue with cells so modified. See, for example,Consigny, U.S. Pat. No. 6,203,487.

The type of cells to be magnetized and delivered will depend upon thespecific condition to be treated, e.g. for repair or therapy of tissuesother than eye, stem cells capable of differentiation into the desiredtissue type, e.g. muscle, skin, nerve, etc., could be delivered. Forexample, photoreceptor or stem cells can be delivered to the macula fortreatment of Retinitis pigmentosa; endothelial cells or stem cells canbe delivered to cornea for treatment of Fuch's endothelial dystrophy orPseudophakic bullous keratopathy; stem cells can be delivered to corneafor treatment of Corneal epithelial disturbance including limbal stemcell deficiency; retinal ganglion cells can be delivered to retina andoptic nerve for treatment of glaucoma or Ischemic optic neuropathy;ocular melanoma could be treated by delivery of a toxin-expressing celltype to the melanoma; Cirrhosis could be treated by delivery ofhepatocytes to the liver; heart failure could be treated by delivery ofcardiac myocytes to the heart; adenocarcinoma, carcinoma, or lymphomacould be treated by delivery of toxin-containing cells to the malignanttumor; skin wounds could be treated by delivery of fibroblasts andepithelial cells to the skin; stroke could be treated by delivery ofstem cells to the brain; and spinal cord injury could be treated bydelivery of stem cells, neurons, or immune cells to the spinal cord.

The methods and compositions disclosed herein can be used to target stemcells, precursor cells, or mature tissue-specific cells, to any site inthe body to facilitate enhanced survival or tissue repair of damaged ordiseased tissues. For example corneas are transplanted for endothelialcell dysfunction; this would provide a technique for replacingendothelial cells without the necessity of a whole transplant operation.The present methods can replace whole tissue transplants, or supplementcurrent stem cell technologies.

This application claims priority to U.S. provisional application No.61/006,861, filed Feb. 4, 2008, which is incorporated herein byreference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: RGCs express trkB. (A) RGCs immunolabeled with anti-trkB (red;DAPI nuclear counterstaining is blue). (B) RGCs were cultured in growthmedia including BDNF, CNTF, insulin and forskolin for 24 hours. 1 μmmagnetic nanoparticles coated with anti-trkB were subsequently added tothe medium for 2 hours, after which time the cultures were washed withfresh medium and the RGCs examined with DIC microscopy.

FIG. 2: A force-distance relationship was constructed based on Stokes'law F=6πηRν, where F is the force due to friction acting on a particleof radius R at a distance d from the tip of the pole piece, traveling atvelocity ν through a fluid viscosity of μ.

DETAILED DESCRIPTION General Methods Cells

Cells for magnetization and use in the method can be obtained accordingto known protocols. For examples below, RGCs were purified tohomogeneity (>99.5%), separating them from all other retinal neurons aswell as all other CNS glial cells (Meyer-Franke et al., 1995; Goldberget al., 2002b; Goldberg et al., 2002a). Purification of RGCs will allowmore rapid identification of nanoparticle binding and endocytosis, andwill allow us to better characterize the force versus axon growth rate.In other examples, CNS glia, both astrocytes and oligodendrocytes(Goldberg et al., 2002b; Goldberg et al., 2002a) can be purified fortesting (Goldberg et al., 2002b; Goldberg et al., 2002a).

Magnetic Nanoparticles

Magnetic nanoparticles in various forms are already in use clinicallyand in research applications without any demonstrated toxicity. Forexample, superparamagnetic particles containing monociystalline ironoxide nanoparticles (MION) of diameters <50 nm have been used as MRIcontrast agents. These particles have demonstrated neurologicnon-toxicity and axonal transport of ferrous-based agents (Neuwelt etal., 1994). Published studies supporting the use of the MRI contrastagent Ferridex (Advanced Magnetics and Berlex Laboratories) have foundno deleterious effects. Furthermore, magnetically directed drugdelivery, using tagged pharmaceuticals in the form of magneticmicrospheres and magnetic polymer carriers, has shown success indelivering anti-neoplastic drugs and radio-isotopes to magneticallytargeted areas in vivo (Schutt et al., 1997; Lubbe et al., 2001).

Coatings

Means for applying the contemplated coatings to magnetic nanoparticlesare well known to those of skill in the art. Commercial kits areavailable having the necessary agents and instructions, for example, asdetailed in the description above and examples below (see, e.g., Example1).

Magnets

Magnets for use in the medical arts and in particular for localizingmagnetic particles in tissue are familiar to those of skill in the art.Suitable magnets are described, for example in Consigny (U.S. Pat. No.6,203,487). The clinical device will include either a superconductingmagnet or fixed/rare earth magnet with sufficient field densityuniformity and magnetic field gradient to direct the cells and hold themin place. Specifics of magnetic field strength will vary by need, suchthat stronger fields/gradients will be used when the magnet is requiredto act at greater distances, and weaker fields/gradients may be usedwhen the magnet can be localized closer to the implanted particlesand/or target tissues. We anticipate directing the cells to the targettissue and then modulating the underlying field to further refine theirmovement and shape the tissue.

EXAMPLES Example 1 Coating Magnetic Nanoparticles for Surface Attachment

For coating various magnetic nanoparticles for surface attachment toneurons, a procedure analogous to that effective for coating 1 μmparticles activated with carboxylic acid (Dynal Biotech, Oslo, NORWAY)or for coating 50 nm particles (e.g. Miltenyi Biotech) with anti-TrkB(BD Bioscience, San Jose, Calif., USA) can be used. The coatingprocedure is performed according to the manufacturer's standardprotocols. Briefly, particles are washed twice with 25 mM MES atapproximate pH6 buffer for approximately 10 min each time. Approximately150 μg of anti trkB in MES buffer is used for functionalizing particles,and slow tilt rotated for approximately 30 min. Then, 0.3 mg of EDC inMES buffer is added, and incubated overnight at 4° C. with tiltrotation. Finally, particles are washed in PBS for four times and PBS isadded to a final 1 mg/ml. We found that 1 μm magnetic particles coatedin this manner can strongly bind to RGCs (FIG. 1). It is expected thatthis and similar protocols can be used to coat magnetic nanoparticlesdown to 25 nm diameter and smaller.

Example 2 Optimal Functionalization (Surface Coating) of CommerciallyAvailable Superparamagnetic Nanoparticles to Maximize Binding to RetinalGanglion Cells

Commercially available surface activated superparamagnetic nanoparticlesas small as 25 nm (MicroMod Partikeltechnologie GmbH, GERMANY) can becoated according to manufacturers' protocols with functional moleculesselected for their ability to strongly and specifically bind neurons.Briefly, tosyl-activated or carboxyl-activated magnetic nanoparticlescan be used for attaching antibodies, proteins and other biomoleculesthat contain primary amino or sulphydryl groups. We will usemanufacturers' suggested protocols for nanoparticle and protein/antibodyconcentrations as a starting point to covalently attach the followingproteins: antibodies to the trkB receptor, antibodies to the surfaceadhesion molecule L1, antibodies to surface integrin receptors, andcholera toxin subunit B, which binds to the GM1 ganglioside on thesurfaces of RGCs and other neurons. We have already successfully shownthat we can functionalize magnetic nanoparticles using these techniques(see above). The coupling of the functional group will be verified bystaining the nanoparticles with fluorescently tagged secondaryantibodies directed against the primary antibody/protein.Non-functionalized magnetic nanoparticles will be used as controls. Wewill confirm that the coating process did not disrupt the ability ofthese antibodies/molecules to bind their targets.

Example 3 Measurement of Binding Specificity of Magnetic Nanoparticlesin Purified and Mixed Cultures

To assay for nanoparticle binding by neurons, retinal ganglion cells(RGCs) can be cultured according to standard protocols (Meyer-Franke etal., 1995; Goldberg et al., 2002b). We will add functionalizednanoparticles generated as described above to the RGCs 2 hours afterplating, leave them for an additional 1 hour at 37° C., and thenexchange the media to remove excess unbound nanoparticles. We will leavethe neurons in culture for 1 hour to 3 days, to examine whether thenanoparticles remain attached with time. At the end of the cultureperiod we will use three techniques to confirm nanoparticle binding: (1)direct visualization using high-magnification microscopy available inthe lab; (2) commercially available iron staining kits (Sigma) in thecase of nanoparticles with exposed iron surfaces; and (3) standardimmunohistochemistry with fluorescent secondary antibodies directedagainst the antibodies/proteins coating the nanoparticles. Using these 3techniques we will estimate at a gross level the amount of nanoparticlebinding by counting nanoparticles or comparing stained cells.

To assay for neuron-specific binding, we will use mixed retinal andcortical cell cultures, both of which we are currently using in the lab.Although most of the studies for initial simplicity will focus on theuse of RGCs, we wish to generate at a minimum some indication that thedata generated for RGCs will be testable more broadly on other CNSneurons. Approximately 2 hours after plating we will add functionalizedmagnetic nanoparticles, as above, and exchange the media after 1 hour at37° C. to remove excess unbound nanoparticles. After 1 hour to 3 days,we will do double immunohistochemistry to determining bindingspecificity, using antibodies against the neuron-specific surfacemolecule thy-1 to identify RGCs or cortical neurons.

Example 4 Measurement of Binding Strength in Varying Magnetic Fields

The magnet will first be calibrated to the magnetic nanoparticles to beused, as nanoparticles in different regions in the dish will experiencedifferent forces. We will initially calibrate two different magnets: (1)a calibrated permanent magnet, and (2) a sharpened magnetized tip. Wewill use uncoated magnetic nanoparticles for calibration by suspendingthem in a high viscosity polydimethylsiloxane solution (PDMS, Sigma). Wewill use 12,000 centistoke PDMS for 1 μm nanoparticles, and 1,000centistoke PDMS solution for nanoparticles smaller than 1 μm. We willplace the permanent magnet in the middle of a 35 mm Petri dish withglass bottom containing magnetic nanoparticles and PDMS solution. Themovement of the nanoparticles towards the magnet will be digitallyrecorded using videomicroscopy, from which we will calculate positionversus time (velocity) of the nanoparticles. We will then plot velocityversus position from magnet to fit a curve, which can then be used toestimate the force versus position (distance) curves based on Stokes'law:

F=6πηRν

wherein F is the force due to friction, η is the fluid viscosity, R isthe nanoparticle radius, and ν is the nanoparticle velocity. This willgive us the force-distance relationship for the specificmagnet/nanoparticle in use (see, e.g. FIG. 2).

To measure binding strength of magnetic nanoparticles to neurons, wewill add functionalized nanoparticles to the RGC cultures as describedabove. We will use a calibrated permanent magnet to apply a known forceto RGC-nanoparticle pairs. We will note whether the nanoparticle wasattached to an axon or the cell body, as binding strength may varyaccording to the cellular site of attachment. By varying the distancebetween the nanoparticle and the magnet, we can vary the applied force,for example to increase the force until the nanoparticles detach fromthe neurons. We will record the time, t, since application of force andthe force at which the nanoparticle detaches from the cell/axon. We willuse this data for statistical analysis of the binding force of thenanoparticle to a cell for a variety of nanoparticle sizes coated withone of the above mentioned molecules.

Using this technique, we have demonstrated that surface activatednanoparticles can be strongly and specifically attached to neurons andother cells. Optimal nanoparticle size should be able to be determinedthrough routine experimentation. Likewise, antibody and protein coatingscan be optimized for individual applications.

Example 5 Delivery of Magnetic Particle-Comprising Cells to a TargetTissue

Magnetic particle-comprising cells as described above can beadministered to the subject by any suitable means known in the art, forexample, by injection (local or systemic), topical application,infusion, etc. It is expected that for applications involving the eye,topical application or local injection will be preferred. Followingadministration of the cells, one or more magnets will be positioned soas to cause the cells to migrate to or remain in or at the desiredtarget tissue. The required strength of the magnet and time periodnecessary for the magnetic force to be applied in order to effect thedesired outcome (in most instances, cells being fixed in or attached tothe target tissue) can be determined by routine experimentation.

Three such examples are offered in detail.

(a) Delivery of donor or autologous corneal endothelial cells to thecorneal endothelial surface of the patient with inadequately functioningendothelium, as in Fuch's Endothelial Dystrophy or Pseudophakic BullousKeratopathy. Corneal endothelial cells would be isolated from humandonor corneas (Joyce et al., 1990; Joyce et al., 1996; Chen et al.,2001; Joyce, 2003; Joyce and Zhu, 2004; Zhu and Joyce, 2004) or derivedfrom human stem cells in cultures by other technologies (Yokoo et al.,2005; Yamagami et al., 2006). Such corneal endothelial cells would bebound with magnetic nanoparticles, for example 50 nm or 360 nm magneticnanoparticles bought commercially or constructed using published methods(Schroder et al., 1986; Douglas et al., 1987; Sestier et al., 1998;Perrin et al., 1999; McCloskey et al., 2000; Tibbe et al., 2001).Binding of cells to coated nanoparticles would be based on specificantibody-antigen nanoparticle coatings, for example using antibodiesagainst cadherin-11, integrin-beta-1, platelet-derived growth factor1-alpha receptor, or neuropilin-1, all of which are expressed by cornealendothelial cells [our unpublished data]. Such magneticnanoparticle-coated endothelial cells would be injected into theanterior chamber of the eye in a manner that can be done in a clinic,for example with a 30 gauge needle, without a requirement for incisionalsurgery. 10³-10⁶ cells will be delivered by injection in a volume of3-300 μL, but more typically around 10⁴-10⁵ cells in a volume of 50-100μL. A suitable magnet, for example a rare earth magnet of suitablestrength, would be affixed in a patch to the surface of the eye externalto the eyelid centered over the cornea. Over the course of a 1 hour to 7days but more typically 16 hours to 3 days, the magnetic field wouldhelp affix the donor, nanoparticle-bound endothelial cells to thesurface of the host/patient endothelial surface, after which timenatural endothelial cell adhesion would take place, removing the needfor additional magnetic field application. The external magnet would beremoved. With time, the nanoparticles on the surface of the donor cellswould degrade from the surfaces by natural proteolytic mechanisms, andbe washed away in the fluid of the anterior chamber. Their small sizewould allow outflow through the trabecular meshwork and other naturaloutflow pathways without clogging these pathways or elevatingintraocular pressure. The delivery of the magnetic endothelial cells tothe internal corneal surface would allow improved pump function of thecorneal endothelium and removal of fluid (edema) from the cornea. Thecornea would subsequently become more clear, improving vision, and lessedematous, decreasing the pain typically associated with this condition.

(b) Delivery of donor or autologous stem cells, photoreceptors, orretinal pigment epithelial (RPE) cells to the subretinal space inpatients with photoreceptor/RPE dysfunction, as in age-related maculardegeneration or retinitis pigmentosa. As in (a), such cells would bebound with magnetic nanoparticles, and injected subretinally, or perhapsthrough the bloodstream intravenously. Surgical implantation of a magnetor magnetic coil (electromagnet) would precede such injection, forexample by affixing a rare-earth magnet by means of a sutured plate tothe sclera behind the macula using a surgical technique in current usefor the attachment of radioactive plaques in the treatment of ocularmelanoma (Giblin et al., 1989; Shields et al., 1993; Shields et al.,1996; Shields et al., 1997). The magnetic field will cause localizationand retention of the implanted cells at the site of degeneration,typically the macula. After healing and integration processes took hold,the magnet might be surgically removed. Alternatively the magnet couldbe left in place for future, additional cell treatments. The small,nano-scale, surface bound particles would as above degrade from thesurfaces by natural proteolytic mechanisms allowing excretion from theeye. In this treatment paradigm, the delivery of magnetic cells to theposterior aspect of the retina would allow the improved function of thephotoreceptors, enhancing visual acuity and visual field in thesepatients.

(c) Delivery of donor or autologous stem cells or retinal ganglion cellsto the retinal surface, for such diseases as glaucoma or ischemic opticneuropathy, or other optic neuropathies. As in (b), such cells would bebound with magnetic nanoparticles, and injected intravitreally. Ratherthan simply floating around in the vitreous or sinking to the base ofthe eye, a posteriorly place magnet would pull the cells to the surfaceof the retina, perhaps over the macula, or in the retinal region of anacquired visual field deficit. Magnet and placement can be, for exampleas described in (b), above. Sequential localization of the magneticfield towards the head of the optic nerve could pull axons along theirnormal wiring pathways to the brain. As above, the small, nano-scale,surface bound particles would as above degrade from the surfaces bynatural proteolytic mechanisms allowing excretion from the eye. In thetreatment of glaucoma or other optic neuropathies with this version ofthe invention, the improved number of retinal ganglion cells, and theimproved integration of these magnetic cells into the proper location ofthe eye, will allow for improved vision, and will contribute to theneuroprotection of the remaining retinal neurons preventing their celldeath.

References, patents and other publications cited herein are herebyincorporated by reference.

REFERENCES

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1. A method of delivering a cell to a target tissue in an animal, saidmethod comprising, (a) affixing, binding, or attaching at least onemagnetic nanoparticle(s) to the surface of said cell to obtain amagnetic particle-comprising cell; (b) administering said magneticparticle-comprising cell to the animal; and (c) applying a magneticfield to said target tissue under conditions such that said magneticparticle-comprising cell is delivered to specific regions of said targettissue.
 2. The method of claim 1, wherein the magnetic particle isaffixed to the cell surface by means of a cell-surface specific bindingagent, typically an antibody.
 3. The method of claim 1 wherein themagnetic particles have a mean diameter of no more than 500 nm.
 4. Themethod of claim 3 wherein the magnetic particles have a mean diameter ofno more than 200 nm.
 5. The method of claim 1 wherein the magneticparticles comprise iron in any ferromagnetic form.
 6. The method of oneof the preceding claims wherein the magnetic nanoparticles have asurface coating.
 7. The method of claim 6 wherein the surface coating ischemically modified to allow the binding of an antibody, or antibodyfragment, or protein or sugar fragment that binds to cells.
 8. Themethod of one of the preceding claims wherein the magnetic particlecomprising cells are administered by injection or infusion or surfaceapplication.
 9. The method of one of the preceding claims wherein thetarget tissue is an eye.
 10. The method of claim 9 wherein the cell isan ocular or optic nerve cell.
 11. The method of one of the precedingclaims wherein the cell is selected from the group consisting of acorneal cell (epithelial, stromal, or endothelial); a retinal cell (aretinal ganglion cell or photoreceptor or other retinal neuron); aretinal glial cell (a retinal muller glial cell or retinal astrocyte orother); a retinal endothelial cell or pericyte; a retinal progenitorcell; a retinal stem cell; an optic nerve glial cell (an astrocyte,oligodendrocyte, microglial cell, or one of their precursors); or a stemor progenitor cell capable of either differentiating into an ocular oroptic nerve cell or supporting the survival or growth or normal functionof an ocular or optic nerve cell.
 12. The method of one of the precedingclaims wherein the animal is a mammal.
 13. The method of claim 12wherein the mammal is a human.
 14. A normal or genetically modified cellhaving a magnetic nanoparticle bound (affixed) to its surface covalentlyor by antibody-antigen linkage.
 15. The cell of claim 14 which is anocular cell or stem cell.
 16. The cell of claim 14 that is selected fromthe group consisting of a corneal cell; a retinal cell; a retinal glialcell; a retinal endothelial cell or pericyte; a retinal progenitor cell;a retinal stem cell; an optic nerve glial bell; and a stem or progenitorcell capable of either differentiating into an ocular or optic nervecell or supporting the survival or growth or normal function of anocular or optic nerve cell.
 17. The cell of claim 16 that is a cornealcell selected from the group consisting of an epithelial cell, a stromalcell and an endothelial cell.
 18. The cell of claim 16 that is a retinalcell selected from the group consisting of a retinal ganglion cell, aphotoreceptor cell and a retinal neuron.
 19. The cell of claim 16 thatis a retinal glial cell selected from the group consisting of a retinalmuller glial cell and a retinal astrocyte.
 20. The cell of claim 16 thatis an optic nerve glial cell selected from the group consisting of anastrocyte, an oligodendrocyte, a microglial cell, and precursorsthereof.
 21. The cell of one of claims 14-20 wherein the magneticparticle is bound via an antibody-antigen complex.
 22. The cell of claim21 wherein the magnetic particle has a diameter of less than 500 nm. 23.The cell of claim 22 wherein the magnetic particle has a diameter ofless than 200 nm.
 24. The cell of one of claims 14-23 wherein themagnetic particle comprises iron in any ferromagnetic form, with orwithout an inert surface coating, with its surface chemically modifiedto allow the binding of an antibody, or antibody fragment, or protein orsugar fragment that binds to cells.
 25. A method of treating a diseaseor disorder selected from the group consisting of macular degeneration,retinitis pigmentosa, and other retinal degenerations; glaucoma,ischemic optic neuropathy, and other retinal ganglion cell and opticnerve degenerations; Fuch's endothelial dystrophy, pseudophakic bullouskerathopathy, and other corneal endothelial cell degenerations; cornealepithelial cell degeneration, corneal limbal cell deficiency, and othercorneal epithelial cell degenerations; ocular melanoma, lymphoma, andother ocular cancers; said method comprising (a) administering amagnetic nanoparticle-comprising cell of one of claims 14-24 to theanimal; and (b) applying a magnetic field to a target tissue underconditions such that said magnetic nanoparticle-comprising cell isdelivered to specific regions of said target tissue; thereby alleviatingsymptoms of the disease or disorder.
 26. The method of claim 25 whereinthe animal is a human and the target tissue is an eye.
 27. The method ofclaim 26 wherein the disease is macular degeneration, the nanoparticlecomprising cell is a photoreceptor, retinal pigment epithelial cell, orstem cell, and the target tissue is the macula.